Diagnostic technique for disturbed thrombocyte aggregation accompanying mucoviscidosis in children

FIELD: medicine.

SUBSTANCE: invention represents a diagnostic technique for the disturbed thrombocyte aggregation accompanying mucoviscidosis in children involving a thrombocyte aggregation test using the Multiplate aggregometer inducers. Trays with a magnetic mixer and electrodes are added with NaCl 400 mcl at 37°C and immediately added with whole blood 400 mcl from a hirudin test tube, incubated in the chamber for two minutes; the tray is added with 30 mcl of an aggregation inducer specified in a group: soluble thrombin receptor - peptid-6, adenosine diphosphate, arachidonic acid. The thrombocyte aggregation rate is displayed on the screen in the form of a curve, and the sub-curve area U is automatically calculated; the sub-curve area U shows the thrombocyte aggregation state as compared to reference values in the group of healthy children; if the threshold area U has appeared to exceed the reference, the thrombocyte hyperaggregation, while the threshold area U being less than the reference, the thrombocyte hypoaggregation is stated.

EFFECT: invention provides the timely diagnosis of microcirculatory disorders accompanying mucoviscidosis.

2 ex, 1 dwg

 

The invention relates to medicine, namely to Hematology for the diagnosis of conditions with increased thrombogenic risk and can be used to detect hidden violations of platelet hemostasis at the microcirculatory level in children with cystic fibrosis.

According to genetic studies, the prevalence of cystic fibrosis (MB) in Russia is more than 1 in 10,000 newborns. Given the characteristics and the clinical course of cystic fibrosis in children and are more likely to develop various complications, the urgent task is the study of platelet hemostasis. Special attention should be paid to the ratio of inflammatory changes in inflammation (in loko morbi) and systemic inflammation in chronic obstructive pathology, as the inflammatory response with increased activity of Pro-inflammatory factors in bronchopulmonary system can move up to the level of systemic blood flow. Data pathological changes are characterized by subclinical chronic inflammatory reaction with low activity, which is manifested by disturbances in the microvasculature.

There is a method of study of intravascular platelet aggregation in vitro. The essence of the method lies in the fact that produce blood, stabilize it, the separation of the plasma is at and erythrocytes with obtaining platelet-rich plasma, disaggregation of platelets, the analysis of the obtained data, which is judged on the degree of aggregation, stabilization of blood after collection carried out by the sodium citrate 3.8% in the ratio of 9:1, the separation of blood with obtaining platelet-rich plasma is conducted by centrifugation, prior to the introduction of desegregate part of the platelet-rich plasma get depleted, for samples of each patient determine the extreme values of transmittance, depleted platelet-plasma - 100% light transmission, platelet-rich plasma - 0%, after the introduction of disaggregation off curve disaggregation, which captures the value of the maximum radius value of disaggregation, which is judged on the degree of intravascular platelet aggregation (Patent RF №2102754).

There is a method of assessing the influence of platelet aggregation factors of hemostasis when performing clinical and functional studies, including the study of blood plasma, with platelet-rich plasma is divided into two aliquots, the first and second and only in the first aliquot perform irreversible platelet aggregation under the influence of the inductor, the modified first aliquot and the initial second synchronous centrifuged receive the first and second case, platelet-poor plasma, separately determined in the first and second aliquot contained in poor what rombaldoni plasma identical factors calculate the difference between them and the consumption and release of these factors assess the degree of platelet aggregation factors of hemostasis (RF Patent No. 2179318).

There is a method of study of platelet aggregation, namely, that of platelet-rich plasma is placed in a chamber rheoscope, mix and measure the integral optical density of all units, the integral optical density of all of the aggregates formed by the interaction of cells, the integral optical density of each unit at the end of the aggregation process and calculate the index R agregatirovanija by the formula: R=K/N×100%, where K is the number of large units with integral optical density of more than 151 srvc. units; M is the total number of units with integral optical density greater than 10 services. units; 100% is the factor that determines the percentage relationship of K/N (RF Patent No. 2421724).

The known method of detecting violations of platelet aggregation, for which take blood, secrete platelet-rich plasma and put on a glass slide, where they test for platelet aggregation with multiple inductors. Visual assessment of platelet aggregation under these conditions allows a small volume of 0.02 ml) of plasma and a small amount of inductors to simulate real-world conditions krovat the ka when using the minimum vzaimootmenyaemy concentrations of inducers with the mandatory inclusion in the combination of collagen and calculates an average value of the induction of aggregation by collagen, largest of which is judged on thin violations of the aggregation capacity of platelets. (Patent RF №2393485). The way we have chosen as a prototype.

The disadvantage of this method is the use of blood plasma, obtained by centrifugation, separation of plasma, the calculation of the coefficients of induction of aggregation.

The objective of the invention is to develop a method for the diagnosis of disorders of platelet aggregation to identify violations platelet hemostasis in cystic fibrosis children.

The technical result of the implementation of the method is that the diagnosis of disorders of platelet aggregation allows you to notice any change in the microvasculature in cystic fibrosis and conduct therapeutic correction of these violations.

The method consists in the fact that conduct tests on platelet aggregation with inductors in whole blood in children with cystic fibrosis, as well as the aggregation inductors are used: soluble thrombin receptor-peptide-6 (TRAP-6), the solution by acid (ADP) or arachidonic acid.

The method is as follows.

Platelet aggregation is determined in whole blood on aggregometry "Multiplate" (VD, Germany). Blood should be used within 30 minutes after intake. No reservation is centrifuged the e and secretion of platelet-rich plasma. In a cuvette with a magnetic stirrer and electrode add 400 ál NaCl(3 mmol/l) at 37°C and then add 400 ál of whole blood from a test tube with hirudin. Incubated in the chamber of the device for two minutes, then add in the cuvette 30 μl of aggregation inducer selected from the group: peptide -6 (TRAP-6), adenosine diphosphate (ADP) or arachidonic acid. In each cuvette add one inductor and incubated for 6 minutes. The rate of platelet aggregation assessed on the screen of the device along the curve aggregation and area under the curve (U), which is calculated by aggregometry automatically (Fig.1). Measurement of platelet aggregation spend impedance method. Impedance (eng. impedance, from lat. impedio - inhibit) - method study of the electrical double layer, mechanism and kinetics of the processes at the electrode - electrolyte interface, which is based on the measurement of impedance - the impedance of the electrochemical cell and the dependence of this resistance on the frequency of the alternating current. The cell includes researched and auxiliary electrodes between which the electrolyte.

The device draws a curve aggregation on the graph on the Y axis is the rate of formation of aggregates, and on the X-axis the time. The value of the area under the curve U is judged on the state of aggregation of platelets. For each activator: peptide -6(TRAP-6), arachidonic acid, adenosine diphosphate (ADP) under the tsya separate graphics.

Then, assess the area under the curve U in comparison with the reference intervals for different age groups defined in healthy children:

0.1 to 12 months.: (TRAP-6) 32-78 U; ADP 24-83 U; arachidonic acid 27-73 U;

1,1-4 years: (TRAP-6) 43-90 U; ADP 32-75 U; arachidonic acid 45-90 U;

5-9 years: (TRAP-6) 58-95 U; ADP 35-74 U; arachidonic acid 56-90 U;

10-18 years: (TRAP-6) 54-97 U; ADP 28-74 U; arachidonic acid 52-88 U.

The U value higher than the maximum reference value corresponding age group demonstrates hyperaggregation platelets with this inducer of aggregation.

The U value is below the threshold, the corresponding age group demonstrates hypogravity platelets with the inductor.

On the basis of the obtained data it can be concluded that the changes occurring in the body of a sick child with cystic fibrosis can affect the aggregation capacity of platelets. In case of detection of violations of aggregation correction of dysfunction platelet hemostasis purpose, in addition to the basic therapy, drugs with angioprotective action: origin, endotelon, angiojet, arginine and antiplatelet properties: pentoxifylline, cardiomagnyl, plaice.

These laboratory tests can be used in the diagnosis of platelet disorders C is s hemostasis in cystic fibrosis children. We studied 50 patients with cystic fibrosis with pulmonary gastrointestinal form ages 3 months and up to 15 years. The control group consisted of 40 healthy children of the same age. The diagnosis of cystic fibrosis was staged on the basis of positive sweat test (>60 mmol/l concentrations of sodium and chloride in sweat fluid) and confirmed by genetic studies.

The invention meets the criterion of "novelty", because currently there is no way to detect changes in the aggregation properties of platelets with cystic fibrosis, and therefore, prevention of disturbances of microcirculation, ischemic changes in the focus of inflammation.

We have shown that routine methods, namely the definition of the standard indicators of the General analysis of blood, coagulation, often does not give us a full picture of the processes occurring at the microcirculatory level.

Clinical example 1.

Girl, J. E., 3 years was in the Department of pulmonology and Allergology with a diagnosis of Testoviron of the pancreas (mucoviscidosis E 84.0), genotype 218ins A/ 2143 delT, pulmonary, intestinal form, severe. Chronic diffuse bronchitis. NAM 1 degree. Complaint: on wet cough productive cough with a moderate number (on average, 50 ml) yellow-gray sputum, poor weight gain. Sick 2 months, when there was a dry joke is obrazny cough with viscous sputum and lumps of thick green mucus. In the survey by place of residence suspected diagnosis of cystic fibrosis, which was confirmed by laboratory tests (chloride sweat 147 mmol/l). A further survey was carried out in the ncla Russian Academy of medical Sciences Department of pulmonology and Allergology. The diagnosis is confirmed by genetic studies. When viewed noteworthy low physical development of the child. Weight 11,8 kg; increase of 92.5 see At laboratory examination the following indicators of a blood: erythrocytes 4,82x1012/l, hemoglobin 127 g/l, leucocytes 6,12xl0%; erythrocyte sedimentation rate of 7 mm/h, platelets 448x109/liter coagulogram violations have been found. Conducted tests to assess the aggregation of platelets. Revealed the following values: peptide TRAP-6 - 27 U (reference values 43-90 U); ADP - 17 U (reference values 32-75 U); arachidonic acid 12 U (reference values 45-90 U), which shows that hypogravity platelets. These changes characterize a violation of platelet aggregation in the microcirculation level under normal number of platelets in the peripheral blood. On the basis of the received data to the basic therapy was added menadione, calcium and magnesium. After 14 days of repeated study of platelet aggregation. The obtained data aggregation: with TRAP-6 - 84 U; ADP - 27 U; arachidonic acid - 61 U (significance level was p<0,05). On the fo is e therapy was noted positive dynamics of the broncho-pulmonary system, perhaps due to the correction of disorders of platelet aggregation in the microcirculation level.

Clinical example 2.

Girl A., 7 years was in the Department of pulmonology and Allergology diagnosed with:

cystic fibrosis, mixed, severe. Chronic diffuse bronchitis.Common cylindrical bronchiectasis in both lungs. Malnutrition 1-11 degree. The formation of pulmonary heart. Pulmonary hypertension. Secondary angiopathy of the retina. Complaints on frequent paroxysmal cough, difficulty breathing, shortness of breath, recurrent headaches. With the birth of the noted bad weight gain, frequent pneumonia. Diagnosed in 3 months, confirmed genetically - heterozygote for del F508. Periodically arise hemoptysis. When viewed chest inflated, breathing weakened on both sides, bilateral diffuse dry rales. HR 118 in minutes NPV 22 minutes In the analysis of blood revealed:

erythrocytes 4,95xl012/l, hemoglobin 130 g/l leucocytes 20,7xl09/l, platelets 564x10% ESR 6 mm /h. Platelet aggregation: with TRAP-6 - 125 U (reference values 58-95 U),c ADP-78 U (reference values 35-74 U), and arachidonic acid - 101 U (reference values 56-90 U), which shows that hyperaggregation platelets. On the basis of these indicators to the basic therapy of added metabolic therapy, appointed: carnitine,vitamin E, B and trental with antiagregatini and angioprotective action. After 14 days the aggregation of platelets in the dynamics was: with TRAP-6 - 95 U, and ADP - 59 U.

Thus, noted the positive dynamics in the aggregation properties of platelets, which resulted in the normalization of indicators. The circulation in the blood vessels of various organs associated with the formation of microthrombi in the blood flow and subsidence of these clots in small blood vessels. The child had angiopathy of the retina, which can also serve as a manifestation of metabolic disorders in platelets and rheology at the level of the microvasculature. As a result of insufficient supply of oxygen affected the function of target organs (lungs, bronchi, digestive system) with the formation of microthrombi in the blood flow and subsidence of these clots in small blood vessels.

The study of the aggregation of platelets facilitates timely diagnosis violation of the aggregation properties of platelets and their correction by assigning the necessary preparations in addition to the basic therapy that eliminates the endothelial dysfunction and platelet aggregation at the level of microcirculation and helps patients in conditions of tissue hypoxia.

The advantages of the proposed method for the diagnosis of disorders of platelet link hemo the pelvis in children is the use of whole blood, inductors add in standard doses, use the impedance method of measurement, the results of which are presented as area under the curve (U), and not in seconds, which gives the additional ability of platelet aggregation.

Method for the diagnosis of disorders of platelet aggregation in cystic fibrosis children, including test platelet aggregation with inductors, wherein the platelet aggregation is determined in whole blood on aggregometry "Multiplate", while in the cuvette with a magnetic stirrer and electrode add 400 ál of NaCl at 37°C and then add 400 ál of whole blood from a test tube with hirudin, incubated in the chamber of the device for two minutes, then add in the cuvette 30 μl of the inductor aggregation, selected from the group of soluble thrombin receptor - peptide-6, adenosine diphosphate, arachidonic acid, while the rate of platelet aggregation is displayed on the screen of the device in the form of a curve and automatically calculated the area under the curve U, the value of the area under the curve U is judged on the state of aggregation of trombocytes in comparison with the reference values in the group of healthy children and with increasing thresholds square U above reference judge hyperaggregation platelets, and at lower thresholds square U below the reference judge hypogravity platelets.



 

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